Antiosteoporotic agents

ABSTRACT

It is disclosed that compounds of the formula: ##STR1## WHEREIN Y represents (C═O) m  in which m has a value of 0 or 1; n has a value of 0 or 1; and X represents S, NH or O; provided that there is a COOH substituent at the 1, 2 or 3 position relative to the X group; and further provided that when X is NH and n is O, the COOH group cannot be at the 2 position; and further provided that when n is 0, the carboxyl group is attached to the ring containing the X and Y ring members or a nontoxic, pharmaceutically acceptable salt thereof are capable of decreasing the ratio of the rates of bone resportion to bone deposition in a host animal, e.g., in the treatment of osteoporosis.

This invention relates to a process for modifying the balance betweenbone production and bone resorption in a host animal, including man, andmore specifically to novel antiosteoporotic agents.

Osteoporosis is a common condition in adults which is evidenced by adecrease in bone density throughout the body. In fact, both the bonemineral (calcium phosphate called "hydroxyapatite") and the matrix(protein called "collagen") are slowly lost. This condition may begin tooccur in humans as early as age 30. In general, the process is morerapid in women than in men. However, after age 80 there is no sexdifference in the incidence of osteoporosis. In the course of 10 to 20years of bone loss there may be symptoms of back pain and X-ray evidenceof deformation of the back bone. At older ages, the brittleness of thebones becomes evident by the ease in which the hip bone fractures as theresult of a simple fall. Osteoporosis is the most common cause offractures in people over age 45.

Although the cause of osteoporosis is poorly understood, it is believedthat there is an imbalance between bone production and bone resorption(bone breakdown). Bone remains a dynamic tissue throughout the life ofan animal. That is, new bone is continuously being formed and old boneis continuously being resorbed. However, in animals suffering from anosteoporotic condition, bone resorption exceeds bone formation.

A survey indicates that in the United States there may be four millionosteoporotic patients with serious symptoms such as vertebral fractures(D. Whedon, Clinical Endocrinology, II, 349-376 (1968)). Moreover, it isestimated that there are currently another 10 million persons sufferingfrom osteoporosis who have not yet developed symptoms. Various types ofosteoporosis are designated according to special conditions believed tobe causative: senile (aging); post-menopausal (female loss ofestrogenesis); disuse (chronic immobilization); steroid (long termsteroid treatment as in arthritis). Osteoporosis may also be manifestedin dental problems since the jaw bone appears to lose mass more rapidlythan any other bone. Thus, periodontal disease involving a loosening ofthe adult teeth may be an early sign of osteoporosis.

The mechanism of bone loss is at present poorly understood. Moreover,the present methods of treatment are generally unsatisfactory. Theseinclude anabolic agents, various drugs containing phosphorous, VitaminD, estrogens, calcium salts, fluorides and calcitonin.

Anabolic agents and estrogen therapy have been the therapy of choice forosteoporosis in post-menopausal women. Unfortunately, recent studieshave indicated that patients taking estrogens may have an increasedincidence of cancer of the uterus and breast. Thus, the advisability oflonger-term use of such treatments would appear to be questionable.

Physical therapy is another method currently used to treat osteoporosissince immobilization can cause osteoporosis at any age. Thus, manyphysicians believe that exercise and physical therapy can prevent theprogression of the disease in elderly patients. However, physicaltherapy can be harmful for patients with fractures and moreover,overstrenuous exercise can cause fractures in patients with severeosteoporosis.

Other treatments include the administration of a fluoride salt such assodium fluoride which has been shown to promote bone growth clinically,apparently by stimulating collagen synthesis. However, a serious sideeffect is poorly calcified, irregular bone growth. Another treatmentinvolves infusion of calcium and Vitamin D to counteract the deficiencyof calcium or impaired absorption of calcium which is symptomatic insome elderly patients. There is, however, no evidence that a higherintake of calcium will prevent osteoporosis or increase bone mass and itcould increase urinary calcium excretion.

The most promising therapeutic approach to the treatment of osteoporosisis the administration of agents which have been designed to modify thebalance between the rate of bone production and the rate of boneresorption in such a manner that the ratio of the former to the latteris increased, resulting in no net bone loss. After the previouslyoccurred bone losses have been restored, a steady state is reached wherethe rate of bone production and rate of bone resorption are equal. Sucha modification may be effected by stimulating the physiologicalmechanism of bone deposition, i.e., bone formation, or by retarding themechanism of bone resorption, or both. Drugs presently in use or in theexperimental stages for accomplishing these purposes include inorganicphosphate type drugs, calcitonin and mithramycin. However, all of thesedrugs suffer serious drawbacks.

Mithramycin, an antibiotic, has anti-tumor activity together withhypocalcemic activity, i.e., effects a lowering of serum calcium whichin turn is believed to be indicative of a decrease in the relative rateof bone resorption--i.e., bone resportion relative to bone production.Side effects, however, include nausea and renal and hepatic toxicity.Likewise, the inorganic phosphates (called "phosphonates") have sideeffects which include extraskeletal calcification, hypotension and renalfailure, while calcitonin presents an immunological problem by virtue ofits being derived from a foreign, e.g., bovine, etc., source. Thus, noneof the foregoing agents are at present suitable for use in the treatmentof osteoporosis.

The search for new bone resorption and/or bone deposition modifyingagents has, accordingly, continued. Recently, it has been shown that2-thiopene carboxylic acid (hereinafter referred to as 2-TCA) is amarkedly effective hypocalcemic agent (see V. S. Fang et al;Endocrinology, 85, 763, 1969; Fang et al, Science, 172, 163, 1971). Itwas thus theorized that 2-TCA would be capable of reducing the relativerate of bone resorption and this theory has subsequently been verified.However, in certain animal tests 2-TCA has not proven to be effective inpreventing bone loss. As will be hereinafter demonstrated, the presentinvention provides agents which are even more effective than 2-TCA inmodifying the relative rate of bone resorption.

It is an object of this invention to provide a method wherein a hostanimal, including man, suffering from osteoporosis is treated in orderto modify the balance between the rates of bone deposition and boneresorption in said host animal whereby the ratio of the latter to theformer is reduced.

It is another object of this invention to provide a process for thetreatment of a host animal in order to prevent the deterioration ofexisting healthy bone tissues in said host animal.

It is a further object of this invention to provide a process for thetreatment of periodontal disease.

It is yet another object of this invention to provide a treatment forfacilitating the healing of damaged bones.

These and other objects are achieved by the practice of this inventionwhich, briefly, comprises administering to a host animal, including man,a compound of the formula: ##STR2## wherein Y represents (C═O)_(m), mhas a value of 0 or 1; n has a value of 0 or 1 and X represents S, NH orO, provided that there is a COOH substituent at the 1, 2 or 3 positionrelative to the X group; and further provided that when X is NH and n is0, the COOH group cannot be at the 2 position; or their nontoxic,pharmaceutically acceptable salts. It is to be understood that when thevalue of n in the above formula is 0, the COOH group will be attacheddirectly to the heterocyclic ring and that the benzene ring at the leftof the above structure will in all cases remain unsubstituted.

Pharmaceutically acceptable salts include the nontoxic metallic saltssuch as sodium, potassium, calcium and aluminum, the ammonium salt andsubstituted ammonium salts, e.g., salts of such nontoxic amines astrialkylamines including thiethylamine, lysine, procaine, dibenzylamine,N-benzyl-beta-phenethylamine, 1-ephenamine-N,N'-dibenzylethylenediamine,N-(lower)alkylpiperidine, e.g., N-ethylpiperidine.

These compounds are administered in any physiologically acceptablemethod, e.g., orally or parenterally. In physiologically acceptablequantities, e.g., from about 10 μg. to 200 mg. per kg. body weight,preferably from about 0.5 mg. to 200 mg. per kg. body weight, and mostpreferably from about 2 mg. to 50 mg. per kg. of body weight, they arecapable of reducing the relative rate of bone resorption and are thususeful in, for example, the treatment of osteoporosis.

Methods for preparing the following compounds, corresponding to theabove formula, are described in Experiments 1-4.

It will be appreciated from the following formulae that the statement"there is a COOH substituent at the 1, 2 or 3 position relative to the Xgroup", which is used to define the compounds corresponding to the aboveformula, means that the COOH group is situated on either the first,second or third carbon atom adjacent the X group as indicated by thenumbers "1", "2", "2'" and "3'" in the above formula. ##STR3##

The above compounds are hereinafter referred to by the code numbersappearing below the respective formulae.

EXPERIMENT 1

Thionaphthene-2-carboxylic acid (327-9) -- Thionaphthene was treated ina carboxylation reaction in accordance with the procedure described byD. A. Shirley and M. D. Cameron, in J. Am. Chem. Soc., 72, 2788, 1950using N-butyllithium in ether, followed by carbonation with dry ice, andhydrolysis to the acid. The product had a melting point of 238° - 239°C.

Analysis: Calc'd: C, 60.66; H, 3.39; S, 17.99; Found: C, 60.75; H, 3.55;S, 18.06.

EXPERIMENT 2

Thionaphthene-3-carboxylic acid (371-25) -- This compound was preparedfrom 3-bromothionaphthene by a Grignard reaction utilizing magnesium inether, followed by carbonation with dry ice, and hydrolysis to the acidin accordance with the procedure described by Crook, J. Am. Chem. Soc.,1937 (1967). The product had a melting point of 178° - 179° C.

Analysis: Calc'd: C, 60.66; H, 3.39; S, 17.99; Found: C, 60.60; H, 3.35;S, 17.83.

EXPERIMENT 3

Dibenzothiophene-4-carboxylic acid (356-3) -- This compound was preparedby the carboxylation of the lithium salt of dibenzothiophene inaccordance with the procedure described by Gilman et al in J. Am. Chem.Soc., 61, 109, 1939 and Gilman et al in J. Am. Chem. Soc., 67, 1479,1945. The product had a melting point of 253° - 255° C.

Analysis: Calc'd: C, 68.40; H, 3.53; Found: C, 68.07; H, 3.57.

EXPERIMENT 4

Thioxanthene-9-one-4-carboxylic acid (356-19) -- This compound wasprepared by adding dropwise a solution of thioxanthene in n-butyletherto a solution of n-butyllithium in hexane. The intermediate product wascarbonated with dry ice and acidified with hydrochloric acid. The finalproduct had a melting point of greater than 300° C.

Analysis: Calc'd: C, 63.93; H, 3.27; S, 13.12; Found: C, 64.53; H, 3.26;S, 13.39.

Indole-2-carboxylic acid (371-59) is commercially available.

The following examples serve to illustrate the effectiveness of theantiosteoporotic agents of the present invention:

EXAMPLE 1

It has recently been shown that the bone remodeling hormones, e.g.,thyrocalcitonin (TCT), is capable of reducing bone resorption rates.Accompanying this reduction, physiologically, is an increase in theproduction of cyclic adenosine-3',5'-monophosphate (c-AMP). Accordingly,the effectiveness of any given bone resorption modifying agent may bedetermined by measuring the effect of that particular agent on theproduction of c-AMP, utilizing isolated bone cells as the test mediumaccording to the methods of Rodan et al, J.B.C., Vol. 429, p. 306, 1974;Rodan et al, Science, Vol. 189, p. 467, 1975. In accordance with thesemethods, compounds of the present invention were tested at aconcentration of 2 μg. per ml. of water, at a pH of 7.4 buffered. Thescreening method measured accumulation of c-AMP in bone cells isolatedfrom newborn rats' calvaria. Theophylline was used in the system as aphosphodiesterase inhibitor, since phosphodiesterase would otherwisedestroy c-AMP. C-AMP was measured by radioimmunoassay. The results setforth in Table 1 are presented in the form of percent activity which wascalculated by comparing the production of c-AMP produced by the compoundbeing tested relative to the c-AMP produced by TCT. Specifically, theresults were calculated according to the formula:

                  TABLE 1                                                         ______________________________________                                        Compound         Percent Activity                                             ______________________________________                                        371-25           200                                                          356-3            150                                                          371-59           100                                                          327-9             80                                                          356-19            56                                                          ______________________________________                                    

wherein the term "control" refers to bone cells which were treated withno compound. As can be seen from the above equation, when TCT is used asthe test compound, "Percent Activity" will have a value of 100. Thus,the "Percent Activity" value for each compound is actually a comparisonof the activity of the test compound with that of TCT. ##EQU1##

In summary, the foregoing data demonstrate the effectiveness ofantiosteoporotic agents of the present invention in stimulating theproduction of c-AMP. It can be seen that the most effective compound wastwice as effective as TCT in stimulating the production of c-AMP whilethe least effective compound was more than half as effective as TCT. Inview of the correlation between stimulation of c-AMP production and boneresorption rates, it can be seen that the foregoing compounds aresurprisingly effective bone resorption modifiers.

EXAMPLE 2

The procedure of Example 1 was repeated utilizing 2-TCA as the bestcompound. Results were calculated in the same manner as above. It wasfound that 2-TCA exhibited a percent activity of 30.

In comparison, compounds of the present invention, as shown in Example1, have percent activity ranging from 200 to 56. It has thus beendemonstrated that compounds useful in this invention are more effectivethan 2-TCA in stimulating c-AMP production.

EXAMPLE 3

The following bone tissue culture investigations were performedutilizing compounds 327-9 and 356-3 to determine the in vitro activityof these compounds. Newborn rat tibiae were incubated in culture mediain accordance with the procedure described by Rodan et al in CalcifiedTissue Res., Vol. 18, p. 125, 1975, with and without the drugs at aconcentration of 20 mg. of drug/ml. of water at a pH of 7.4 for eightdays. Among the parameters measured, only the calcium content was foundto differ significantly between the treated bone tissue and thecontralateral control (untreated bone tissue). The results are presentedin the following table:

    ______________________________________                                                 Treated      Contralateral                                           Drug     Tibia (grams)                                                                              Tibia (grams)                                                                              P*                                         ______________________________________                                        356-3    12.44 ± 0.74                                                                            11.75 ± 0.16                                                                            0.1                                        327-9    12.92 ± 0.08                                                                            11.71 ± 0.05                                                                            0.05                                       Control  12.05 ± 0.08                                                                            11.93 ± 0.64                                                                            NS**                                       ______________________________________                                         *Proportion (estimated on the basis of students' tests for comparison of      paired values).                                                               **Not significant.                                                       

It can be seen that bone tissue treated with the anti-osteoporoticagents of this invention (356-3 and 327-9) actually had a larger calciumcontent than untreated bone tissues. Accordingly, it will be appreciatedthat treatment of bone tissues in accordance with the methods of thepresent invention results in a decrease of the ratio of the rates ofbone resportion to bone deposition.

EXAMPLE 4

The procedure of Example 3 was repeated utilizing 2-TCA as the testcompound. As can be seen from the results below, 2-TCA had nosignificant effect on calcium content.

    ______________________________________                                                 Treated      Contralateral                                           Drug     Tibia        Tibia        P*                                         ______________________________________                                        2-TCA    12.37 ± 0.63                                                                            12.15 ± 0.47                                                                            NS                                         ______________________________________                                         *Proportion (estimated on the basis of students' tests for comparison of      paired values).                                                          

EXAMPLE 5

The in vitro tissue culture conditions in Examples 1-4, are largelyunphysiological due to the lack of vascularization, lack of mechanicalstress, reduced rate of bone remodeling, lack of integration into thefeedback control loops of the organism and the possible absence from themedia of other necessary but unknown factors. Therefore, compound 327-9was tested in vivo for its ability to prevent immobilizationosteoporosis. Rats (150-180 g.) were utilized as the subjects in thisexperiment. The triceps tibial insertion (knee cap tendons) were severedin one rear leg of each rat. One group of the rats was treated for threedays with 327-9 (1 mg./day by s.c. insertion) while the control groupwas not treated with the drug. The animals were sacrificed six daysafter severance of the tendons. They were then dissected and both tibiae(immobilized and non-immobilized) were removed from each animal. Thetibiae were weighed and analyzed for calcium and phosphate content. Theresults obtained for the disuse (immobilized) tibiae were compared tothe results obtained for the contralateral (non-immobilized) tibiae foreach group. As seen in the following table, significant loss of weightand calcium occurred in the immobilized tibiae in the control groupwhile this loss did not occur in the animals treated with 327-9.Moreover, the ratio of calcium to phosphate (an index of the degree ofbone mineralization) was significantly higher in both tibiae bones ofthe animals treated with 327-9. Thus, it can be seen that theadministration of 327-9 prevented the onset of osteoporotic decay ofhealthy bone tissue.

    ______________________________________                                        Wet Weight (gram)                                                                     Disuse     Contralateral                                                                            Ratio    P*                                     ______________________________________                                        Control 0.380 ± 0.019                                                                         0.404 ± 0.014                                                                         0.94 ± 0.04                                                                         0.01                                   327-9                                                                         (1 mg/d)                                                                              0.394 ± 0.06                                                                          0.390 ± 0.050                                                                         1.01 ± 0.05                                                                         N.S.                                   p**     0.01       N.S.       0.01                                            Calcium Content (mmoles/bone)                                                         Disuse       Contralateral                                                                              p*                                          ______________________________________                                        Control 0.992 ± 0.110                                                                           1.103 ± 0.050                                                                           0.01                                        327-9                                                                         (1 mg/d)                                                                              1.181 ± 0.060                                                                           1.121 ± 0.090                                                                           N.S.                                        p**     0.01         N.S.                                                     Phosphate Content (mmoles/bone)                                                       Disuse       Contralateral                                                                              p*                                          ______________________________________                                        Control 0.639 ± 0.050                                                                           0.647 ± 0.048                                                                           N.S.                                        327-9                                                                         (1 mg/d)                                                                              0.657 ± 0.060                                                                           0.612 ± 0.043                                                                           0.05 p 0.10                                 p**                                                                           Calcium/Phosphate Ratio                                                               Disuse       Contralateral                                                                              p*                                          ______________________________________                                        Control 1.050 ± 0.070                                                                           1.680 ± 0.160                                                                           0.02                                        327-9                                                                         (1 mg/d)                                                                              1.810 ± 0.210                                                                           1.830 ± 0.120                                                                           N.S.                                        p**     0.01         0.05                                                     ______________________________________                                         *Proportion (estimated on the basis of students' tests for comparison of      paired values.                                                                **Estimated on the basis of students tests for comparison of population       means.                                                                        N.S., not significant.                                                   

EXAMPLE 6

In this example, the dose of 327-9 was reduced to 200 μg./day. The drugwas administered for six days and the rats were sacrificed nine daysafter the beginning of the experiment. Weight, calcium and phosphatecontent and the density of 110° C.-dried bones were measured. Theresults summarized in the following table show that at this level oftreatment the drug did not prevent weight loss or a drop in the specificdensity of the bone. It was, however, effective in the prevention ofcalcium loss.

    ______________________________________                                        Dry Weight (gram/bone)                                                                Disuse       Contralateral                                                                              P*                                          ______________________________________                                        Control 0.181 ± 0.011                                                                           0.187 ± 0.013                                                                           0.001                                       327-9                                                                         (200 mg/d)                                                                            1.176 ± 0.010                                                                           0.189 ± 0.008                                                                           0.001                                       Density (determined in mineral oil,                                           calculated relative to water at 4° C.)                                         Disuse       Contralateral                                                                              P*                                          ______________________________________                                        Control 1.066 ± 0.023                                                                           1.119 ± 0.044                                                                           0.01                                        327-9                                                                         (200 mg/d)                                                                            1.053 ± 0.034                                                                           1.104 ± 0.037                                                                           0.01                                        Calcium Content (mmoles/bone)                                                         Disuse       Contralateral                                                                              P*                                          ______________________________________                                        Control 0.926 ± 0.091                                                                           0.981 ± 0.059                                                                           0.02                                        327-9                                                                         (200 mg/d)                                                                            0.936 ± 0.058                                                                           0.936 ± 0.108                                                                           N.S.                                        Phosphate Content (mmoles/bone)                                                       Disuse       Contralateral                                                                              P*                                          ______________________________________                                        Control 0.624 ± 0.089                                                                           0.645 ± 0.089                                                                           N.S.                                        327-9                                                                         (299 mg/d)                                                                            0.638 ± 0.052                                                                           0.632 ± 0.100                                                                           N.S.                                        ______________________________________                                         *Proportion (estimated on the basis of students' tests for comparison of      paired values).                                                               N.S., not significant.                                                   

EXAMPLE 7

In this example, the effectiveness of the drugs in preventing the lossof tensile strength was measured. One leg of each animal was immobilizedas described above. The drugs were injected for 10 days (s.c. 1mg./day). The tibiae were dissected and tested for tensile strength, asdetermined by breaking loads. Results are presented in the followingtable:

    ______________________________________                                        Breaking Loads (lbs.)                                                                  Immobilized tibia                                                                          Contralateral tibia                                     ______________________________________                                        Control rats                                                                                10.0 ± 2.3 (SD)                                                                        15.0 ± 4.1                                       327-9 treated                                                                            15.9 ± 7.0  17.6 ± 8.9                                       356-3 treated                                                                            16.9 ± 5.2  16.0 ± 4.9                                       ______________________________________                                    

It can be seen that in the control animals, immobilization caused asignificant decrease in the tensile strength of the tibia whereas in theanimals treated with agents of the invention, immobilization had nosignificant effect. Accordingly, it can be seen that the agents used inthe process of the present invention were effective in preventing lossof tensile strength in the healthy bone tissue of the treated hostanimals.

We claim:
 1. A process for the treatment of a host animal in order tomodify the balance between the rate of bone resorption and the rate ofbone deposition in said host animal whereby the ratio of said rate ofbone resorption to said rate of bone deposition is reduced comprisingadministering to said host animal an amount sufficient to modify saidbalance and reduce said ratio, of a compound of the formula: ##STR4##wherein n has a value of 0 or 1; provided that there is a COOHsubstituent at the 1, 2, 2' or 3' position relative to the S group; andfurther provided that when n is 0, the carboxyl group is attached to thering containing the S group; or a nontoxic, pharmaceutically acceptablesalt thereof.
 2. A process in accordance with claim 1, wherein saidcompound is administered in an amount of from 10 m.μ. to 200 m.g. perk.g. of body weight.
 3. A process in accordance with claim 1, whereinsaid compound is administered in an amount of from 0.5 m.g. to 200 m.g.per k.g. of body weight.
 4. A process in accordance with claim 1,wherein said compound is administered in an amount of from 2 m.g. to 50m.g. per k.g. of body weight.
 5. A process in accordance with claim 1,wherein said compound has the formula: ##STR5##
 6. A process inaccordance with claim 1, wherein said compound has the formula: ##STR6##7. A process in accordance with claim 1, wherein said compound has theformula: ##STR7##
 8. A process in accordance with claim 1, wherein saidhost animal is treated for osteoporosis.
 9. A process in accordance withclaim 1, wherein said host animal is being treated for periodontaldisease.
 10. A process in accordance with claim 1, wherein said hostanimal is treated to prevent the deterioration of healthy bone tissue.